The invention relates to relatively short peptides (termed conantokins herein), about 10-30 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available-peptides, and which include preferably one to two or more xcex3-carboxyglutamic acid residues. The conantokins are useful for the treatment of neurologic and psychiatric disorders, such as anticonvulsant agents, as neuroprotective agents or for the management of pain.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
The predatory cone snails (Conus) have developed a unique biological strategy. Their venom contains relatively small peptides that are targeted to various neuromuscular receptors and may be equivalent in their pharmacological diversity to the alkaloids of plants or secondary metabolites of microorganisms. Many of these peptides are among the smallest nucleic acid-encoded translation products having defined conformations, and as such, they are somewhat unusual. Peptides in this size range normally equilibrate among many conformations. Proteins having a fixed conformation are generally much larger.
The cone snails that produce these peptides are a large genus of venomous gastropods comprising approximately 500 species. All cone snail species are predators that inject venom to capture prey, and the spectrum of animals that the genus as a whole can envenomate is broad. A wide variety of hunting strategies are used, however, every Conus species uses fundamentally the same basic pattern of envenomation.
Several peptides isolated from Conus venoms have been characterized. These include the xcex1-, xcexc- and xcfx89-conotoxins which target nicotinic acetylcholine receptors, muscle sodium channels, and neuronal calcium channels, respectively (Olivera et al., 1985). Conopressins, which are vasopressin analogs, have also been identified (Cruz et al., 1987). In addition, peptides named conantokins have been isolated from Conus geographuis and Conus tulipa (Mena et al., 1990; Haack et al., 1990). These peptides have unusual age-dependent physiological effects: they induce a sleep-like state in mice younger than two weeks and hyperactive behavior in mice older than 3 weeks (Haack et al., 1990).
The conantokins are structurally unique. In contrast to the well characterized conotoxins from Conus venoms, most conantokins do not contain disulfide bonds. However, they contain 4-5 residues of the unusual modified amino acid xcex3-carboxyglutamic acid. The occurrence of this modified amino acid, which is derived post-translationally from glutamate in a vitamin K-dependent reaction, was unprecedented in a neuropeptide. It has been established that the conantokins have N-methyl-D-aspartate (NMDA) antagonist activity, and consequently target the NMDA receptor. The conantokins reduce glutamate (or NMDA) mediated increases in intracellular Ca2+ and cGMP without affecting kainate-mediated events (Chandler et al., 1993). Although these peptides have actions through polyamine responses of the NMDA receptors, the neurochemical profile of these polypeptides is distinct from previously described noncompetitive NMDA antagonists (Skolnick et al., 1992).
The previously identified conantokins are Conantokin G (Con G) and Conantokin T (Con T). Con G has the formula Gly-Glu-Xaa1-Xaa1-Leu-Gln-Xaa2-Asn-Gln-Xaa2-Leu-Ile-Arg-Xaa2-Lys-Ser-Asn (SEQ ID NO:1), wherein Xaa1 and Xaa2 are xcex3-carboxyglutamic acid (Gla). The C-terminus preferably contains an amide group. Con T has the formula Gly-Glu-Xaa1-Xaa1-Tyr-Gln-Lys-Met-Leu-Xaa2-Asn-Leu-Arg-Xaa2-Ala-Glu-Val-Lys-Lys-Asn-Ala (SEQ ID NO:2), wherein Xaa1 and Xaa2 are xcex3-carboxyglutamic acid. The C-terminus preferably contains an amide group. Analogues of Conantokin G have been synthesized and analyzed for their biological activity (Chandler et al., 1993; Zhou et al., 1996). It has been discovered that substitution of the Gla residue at position 4 of Con G destroys its NMDA antagonist properties. Substitution of the Gla residue at position 3 of Con G greatly reduces its NMDA antagonist activity. However, substitutions of the Gla residues at positions 7, 10 and 14 of Con G do not adversely affect potency of the peptide and may even enhance it. (Zhou et al., 1996).
Ischemic damage to the central nervous system (CNS) may result form either global or focal ischemic conditions. Global ischemia occurs under conditions in which blood flow to the entire brain ceases for a period of time, such as may result from cardiac arrest. Focal ischemia occurs under conditions in which a portion of the brain is deprived of its normal blood supply, such as may result from thromboembolytic occlusion of a cerebral vessel, traumatic head or spinal cord injury, edema or brain or spinal cord tumors. Both global and focal ischemic conditions have the potential for widespread neuronal damage, even if the global ischemic condition is transient or the focal condition affects a very limited area.
Epilepsy is a recurrent paroxysmal disorder of cerebral function characterized by sudden brief attacks of altered consciousness, motor activity, sensory phenomena or inappropriate behavior caused by abnormal excessive discharge of cerebral neurons. Convulsive seizures, the most common form of attacks, begin with loss of consciousness and motor control, and tonic or clonic jerking of all extremities but any recurrent seizure pattern may be termed epilepsy. The term primary or idiopathic epilepsy denotes those cases where no cause for the seizures can be identified. Secondary or symptomatic epilepsy designates the disorder when it is associated with such factors as trauma, neoplasm, infection, developmental abnormalities, cerebrovascular disease, or various metabolic conditions. Epileptic seizures are classified as partial seizures (focal, local seizures) or generalized seizures (convulsive or nonconvulsive). Classes of partial seizures include simple partial seizures, complex partial seizures and partial seizures secondarily generalized. Classes of generalized seizures include absence seizures, atypical absence seizures, myoclonic seizures, clonic seizures, tonic seizures, tonic-clonic seizures (grand mal) and atonic seizures. Therapeutics having anticonvulsant properties are used in the treatment of seizures. Most therapeutics used to abolish or attenuate seizures act at least through effects that reduce the spread of excitation from seizure foci and prevent detonation and disruption of function of normal aggregates of neurons. Traditional anticonvulsants that have been utilized include phenytoin, phenobarbital, primidone, carbamazepine, ethosuximide, clonazepam and valproate. Several novel and chemically diverse anticonvulsant medications recently have been approved for marketing, including lamotrigine, feribamate, gabapentin and topiramate. For further details of seizures and their therapy, see Rall and Schleifer (1985) and The Merck Manual (1992).
It has been shown that neurotransmission mediated through the NMDA receptor complex is associated with seizures (Bowyer, 1982; McNamara et al., 1988), ischemic neuronal injury (Simon et al., 1984; Park et al., 1988) and other phenomena including synaptogenesis (Cline et al., 1987), spatial learning (Morris et al., 1986) and long-term potentiation (Collinridge et al., 1983; Harris et al., 1984; Morris et al., 1986). Regulation of these neuronal mechanisms by NMDA-mediated processes may involve activation of a receptor-gated calcium ion channel (Nowak et al., 1984; Mayer et al., 1987; Ascher and Nowak, 1988).
The NMDA channel is regulated by glycine. This amino acid increases NMDA-evoked currents in various tissues [Johnson and Ascher, 1987; Kleckner and Dingledine, 1988] by increasing the opening frequency of the NMDA channel [Johnson and Ascher, 1987]. Thus, NMDA-induced calcium influx and intracellular accumulation may be stimulated by glycine [Reynolds et al., 1987; Wroblewski et al., 1989], which interacts with its own distinct site [Williams et al., 1991]. Furthermore, accumulation of intracellular calcium may be implicated in the aforementioned neuropathologies.
The NMDA receptor is also involved in a broad spectrum of CNS disorders. For example, during brain ischemia caused by stroke or traumatic injury, excessive amounts of the excitatory amino acid glutamate are released from damaged or oxygen deprived neurons. This excess glutamate binds the NMDA receptor which opens the ligand-gated ion channel thereby allowing Ca2+ influx producing a high level of intracellular Ca2+, which activates biochemical cascades resulting in protein, DNA and membrane degradation leading to cell death. This phenomenon, known as excitotoxicity, is also thought to be responsible for the neurological damage associated with other disorders ranging from hypoglycemia and cardiac arrest to epilepsy. In addition, there are reports indicating similar involvement in the chronic neurodegeneration of Huntington""s, Parkinson""s and Alzheimer""s diseases.
Parkinson""s disease is a progressive, neurodegenerative disorder. The etiology of the disorder is unknown in most cases, but has been hypothesized to involve oxidative stress. The underlying neuropathology in Parkinsonian patients is an extensive degenerations of the pigmented dopamine neurons in the substantia nigra. These neurons normally innervate the caudate and putamen nuclei. Their degeneration results in a marked loss of the neurotransmitter dopamine in the caudate and putamen nuclei. This loss of dopamine and its regulation of neurons in the caudate-putamen leads to the bradykinesia, rigidity, and tremor that are the hallmarks of Parkinson""s disease. An animal model has been developed for Parkinson""s disease (Zigmond et al., 1987) and has been used to test agents for anti-Parkinsonian activity (Ungerstedt et al., 1973).
The dopamine precursor, L-Dopa, is the current therapy of choice in treating the symptoms of Parkinson""s disease. However, significant side effects develop with continued use of this drug and with disease progression, making the development of novel therapies important. Recently, antagonists of the NMDA subtype of glutamate receptor have been proposed as potential anti-Parkinsonian agents. (Borman, 1989; Greenamyre and O""Brien, 1991; Olney et al., 1987). In addition, antagonists of NMDA receptors potentiate the behavioral effects of L-Dopa and D1 dopamine receptor stimulation in animal models of Parkinson""s disease. (Starr, 1995). These data suggest that NMDA receptor antagonists may be useful adjuncts to L-Dopa therapy in Parkinson""s disease by decreasing the amount of L-Dopa required and thereby reducing undesirable side effects. In addition, antagonists of NMDA receptors have been shown to attenuate free radical mediated neuronal death. Thus, NMDA receptor antagonists may also prevent further degeneration of dopamine neurons in addition to providing symptomatic relief. Finally, NMDA receptor antagonists have been shown to potentiate the contralateral rotations induced by L-Dopa or D1 dopamine receptor antagonists in the animal model.
Pain, and particularly, persistent pain, is a complex phenomenon involving many interacting components. Numerous studies, however, have demonstrated a role for NMDA receptors in mediating persistent pain, and further that NMDA antagonists are effective in animal models of persistent pain. First, administration of NMDA (the agonist) mimics many of the physiological and behavioral effects of painful stimuli (Chapman et al., 1994; Dougherty and Willis, 1991; Coderre and Melzack, 1992; Malmberg and Yaksh, 1992; Bach et al., 1994; Liu et al., 1997). Second, various classes of NMDA antagonists block the xe2x80x9cwind upxe2x80x9d (progressive augmentation of response caused by repetitive stimulation) of small primary afferent C fibers of the dorsal horn (Davies and Lodge, 1987; Dickenson and Sullivan, 1987; Thompson et al., 1990). Third, release of glutamate and aspartate (agonists at NMDA and non-NMDA glutamatergic receptors) is increased in spinal cord in animal models of persistent pain (Sluka and Westlund, 1992; Malmberg and Yaksh, 1992; Yang et al., 1995). Fourth, NMDA antagonists are effective in animal models of persistent pain (Neugebauer et al., 1993; Coderre, 1993; Coderre and Van Empel, 1994; Yamamoto and Yaksh, 1992; Chaplan et al., 1997; Millan and Seguin, 1994; Rice and McMahon, 1994). Moreover, NMDA antagonists appear to be effective in reducing the tolerance to opioid analgesics seen after chronic administration in animal models of pain (Bilsky et al., 1996; Lufty et al., 1996; Shimoyama et al., 1996; Wong et al., 1996; Elliot et al., 1994; Mao et al., 1994; Dunbar and Yaksh, 1996; Lufty et al., 1995; Trujillo and Akil, 1994; Tiseo et al., 1994; Gutstein and Trujillo, 1993; Kest et al., 1993; Tiseo and Inturrisi, 1993). Finally, severe or prolonged tissue or nerve injury can induce hyperexcitability of dorsal horn neurons of the spinal cord, resulting in persistent pain, an exacerbated response to noxious stimuli (hyperalgesia) and a lowered pain threshold (allodynia). These changes are mediated by NMDA-type glutamate receptors in the spinal cord, whose activation causes release of Substance P, a peptide neurotransmitter made by small-diameter, primary, sensory pain fibres. Injection of NMDA in the cerebrospinal fluid of the rat spinal cord mimicked the changes that occur with persistent injury and produced pain (Liu et al., 1997).
Neuropsychiatric involvement of the NMDA receptor has also been recognized. Blockage of the NMDA receptor Ca2+ channel by the animal anesthetic phencyclidine produces a psychotic state in humans similar to schizophrenia (Johnson et al., 1990). Further, NMDA receptors have also been implicated in certain types of spatial learning (Bliss et al., 1993). In addition, numerous studies have demonstrated a role for NMDA receptors in phenomena associated with addiction to and compulsive use of drugs or ethanol. Furthermore, antagonists of NMDA receptors may be useful for treating addiction-related phenomena such as tolerance, sensitization, physical dependence and craving (for review see, Popik et al., 1995; Spanagel and Zieglgansberger, 1997; Trujillo and Akil, 1995).
There are several lines of evidence which suggest that NMDA antagonists may be useful in the treatment of HIV infection. First, the levels of the neurotoxin and NMDA agonist quinolinic acid are elevated in the cerebrospinal fluid of HIV-positive subjects (Heyes et al., 1989) and in murine retrovirus-induced immunodeficiency syndrome (Sei et al., 1996). Second, the envelope glycoprotein of HIV-1 alters NMDA receptor function (Sweetnam et al., 1993). Thirdly, NMDA antagonists can reduce the effects and neurotoxicity of GP-120 (Muller et al., 1996; Raber et al., 1996; Nishida et al., 1996). Fourth, GP-120 and glutamate act synergistically to produce toxicity in vitro (Lipton et al., 1991). And finally, memantine, an NMDA antagonist, protects against HIV infection in glial cells in vitro (Rytik et al., 1991). For a review of the use of NMDA antagonists in treating HIV infection, see Lipton (1994; 1996).
It is desired to identify additional conantokin peptides and related compounds which target the NMDA receptor. It is further desired to identify compounds which are useful as anticonvulsant, neuroprotective, neuropsychiatric or analgesic agents.
The present invention is directed to conantokin peptides, conantokin peptide derivatives and conantokin peptide chimeras, referred to collectively as conantokins (unless the context dictates otherwise), having 10-30 amino acids, including preferably one to two or more xcex3-carboxyglutamic acid residues. The conantokins are useful for the treatment of neurologic or psychiatric disorders, such as anticonvulsant agents, as neuroprotective agents or for the management of pain. The conantokins are administered to patients as described further below.
More specifically, the present invention is directed to conantokin peptides, which include but are not limited to, G, T, L, R, S1, Oc, Gm, Ca2, Ca1 and Qu. Conantokin G (Con G) has the formula Gly-Glu-Xaa1-Xaa1-Leu-Gln-Xaa2-Asn-Gln-Xaa2-Leu-Ile-Arg-Xaa2-Lys-Ser-Asn (SEQ ID NO:1), wherein Xaa1 and Xaa2 are preferably xcex3-carboxyglutamic acid (Gla). The C-terminus contains a carboxyl or an amide, preferably an amide group. Conantokin T (Con T) has the formula Gly-Glu-Xaa1-Xaa1-Tyr-Gln-Lys-Met-Leu-Xaa2-Asn-Leu-Arg-Xaa2-Ala-Glu-Val-Lys-Lys-Asn-Ala (SEQ ID NO:2), wherein Xaa1 and Xaa2 are preferably xcex3-carboxyglutamic acid. The C-terminus contains a carboxyl or an amide, preferably an amide group. Conatokin L (Con L), has the formula Gly-Glu-Xaa1-Xaa1-Val-Ala-Lys-Met-Ala-Ala-Xaa2-Leu-Ala-Arg-Xaa2-Asp-Ala-Val-Asn (SEQ ID NO:3), wherein Xaa1 and Xaa2 are preferably xcex3-carboxyglutamic acid. The C-terminus contains a carboxyl or an amide, preferably an amide group. Conantokin R (Con R) has the formula: Gly-Glu-Xaa1-Xaa1-Val-Ala-Lys-Met-Ala-Ala-Xaa2-Leu-Ala-Arg-Xaa2-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro (SEQ ID NO:4), wherein Xaa1 and Xaa2 are preferably xcex3-carboxyglutamic acid. The C-terminus contains a carboxyl or an amide, preferably a carboxyl group. The cysteine residues form a disulfide bridge. Conantokin S1 (Con S1) has the formula: Gly-Asp-Xaa1-Xaa1-Tyr-Ser-Lys-Phe-Ile-Xaa2-Arg-Glu-Arg-Xaa2-Ala-Gly-Arg-Leu-Asp-Leu-Ser-Lys-Phe-Pro (SEQ ID NO:5), wherein Xaa1 and Xaa2 are preferably xcex3-carboxyglutamic acid. The C-terminus contains a carboxyl or amide, preferably a carboxyl group. Conantokin Oc (Con Oc) has the formula: Gly-Glu-Xaa1-Xaa1-Tyr-Arg-Lys-Ala-Met-Ala-Xaa1-Leu-Glu-Ala-Lys-Lys-Ala-Gln-Xaa2-Ala-Leu-Lys-Ala (SEQ ID NO:6), wherein Xaa1 and Xaa2 are preferably xcex3-carboxyglutamic acid. The C-terminus contains a carboxyl or amide, preferably an amide group. Conantokin Gm (Con Gm) has the formula: Gly-Ala-Lys-Xaa1-Asp-Arg-Asn-Asn-Ala-Xaa2-Ala-Val-Arg-Xaa2-Arg-Leu-Glu-Glu-Ile(SEQI)NO:7), Xaa1 and Xaa2 are preferably xcex3-carboxyglutamic acid. The C-terminus contains a carboxyl or amide, preferably an amide group. Conantokin Ca2 (Con Ca2) has the formula: Gly-Tyr-Xaa1-Xaa1-Asp-Arg-Xaa2-Ile-Ala-Xaa2-Thr-Val-Arg-Xaa2-Leu-Glu-Glu-Ala (SEQ ID NO:8), wherein Xaa1 and Xaa2 are preferably xcex3-carboxyglutamic acid. The C-terminus contains a carboxyl or amide, preferably an amide group. Conantokin Qu (Con Qu) has the formula: Gly-Tyr-Xaa1-Xaa1-Asp-Arg-Xaa2-Val-Ala-Xaa2-Thr-Val-Arg-Xaa2-Leu-Asp-Ala-Ala (SEQ ID NO:9), wherein Xaa1 and Xaa2 are preferably xcex3-carboxyglutamic acid. The C-terminus contains a carboxyl or amide, preferably an amide group. Conantokin Ca1 (Con Ca1) has the formula: Gly-Asn-Asp-Val-Asp-Arg-Lys-Leu-Ala-Xaa2-Leu-Xaa2-Xaa2-Leu-Tyr-Xaa2-Ile (SEQ ID NO:68), wherein Xaa2 is preferably xcex3-carboxyglutamic acid. The C-terminus contains a carboxyl or amide, preferably an amide group.
The present invention is also directed to conantokin peptide derivatives. Examples of conantokin peptide derivatives include conantokin peptides in which the xcex3-carboxyglutamic acid at the Xaa2 residues in these peptides is replaced by any other amino acids such that their NMDA antagonist activity is not adversely affected. Examples of such replacements include, but are not limited to Ser, Ala, Glu and Tyr. In addition, glutamic acid residues in the peptide can be modified to xcex3-carboxyglutamate residues. Other derivatives are produced by modification of the amino acids within the conantokin structure. Modified amino acids include those which are described in Roberts et al. (1983). Other derivatives include conantokin peptides in which one or more residues have been deleted.
The present invention is also directed to conantokin peptide chimeras. Suitable conantokin chimeras are produced by recombination of different segments of two or more conantokin peptides, conantokin peptide derivatives or a peptide encoded by exon 5 of the NMDA receptor, e.g. Lys-Pro-Gly-Arg-Lys (SEQ ID NO:10) or Lys-Pro-Gly-Arg-Lys-Asn (SEQ ID NO:11).